The effect of mycorrhizae on plant growth and
reproduction varies with soil phosphorus and developmental
stage.

Abstract:

Determining the impact of both environmental variation and
developmental stage on plant-mycorrhizal associations is important, as
both can shift the association along the mutualism-parasitism continuum.
This study examines the effect of phosphorus level on the response of
Allium vineale to mycorrhizae across all plant life stages, including
plant fecundity and the relative allocation of resources to three
different reproductive modes (flowers, asexual underground offsets, and
asexual aerial bulbils). For A. vineale, the impact of mycorrhizae
varies significantly with life stage, as an early growth depression at 1
mo was reversed by 15 mo, resulting in mycorrhizal plants having larger
bulbs over all P levels and producing more bulbils and larger offsets
than nonmycorrhizal plants at lower P levels. However the presence of
mycorrhizae did not affect the relative allocation of resources among
the three reproductive modes. These results emphasize the importance of
long-term studies of plant-mycorrhizal interactions that include
fecundity estimates. In addition, they indicate that spatial variation
in nutrient availability in the field has the potential to shift the
overall effect of mycorrhizae from beneficial to neutral, with greater
benefits found in sites with lower phosphorus levels.

The association between mycorrhizal fungi and plant roots was
important in the evolution of land plants (Brundrett, 2002) and
currently occurs in at least 80% of all plant species (Smith and Read,
1997). Colonization by arbuscular mycorrhizal (AM) fungi can provide
multiple functions, such as increased nutrient uptake, drought
tolerance, and resistance to pathogens (Newsham et al., 1995). Whereas
many studies have shown that AM fungi can increase plant growth rates,
it is widely recognized that there is considerable variation in response
to colonization among plant species (Hart and Klironomos, 2002; Jones
and Smith, 2004). This variation in response has been ascribed to a
range of causes, including abiotic and biotic environmental factors,
differential effects of colonization over the life cycle of the plant,
and specificity in the association between the fungal-plant partners
(Johnson et al., 1997; Jones and Smith, 2004).

Environmental factors such as light and nutrient availability may
shift the plant-mycorrhizal balance from beneficial, to neutral, or even
negative (Johnson et al., 1997). For example, under high phosphorus
conditions plant biomass may be reduced in the presence of mycorrhizal
fungi (e.g., Buwalda and Goh, 1982; Peng et al., 1993; Olsen et al.,
1999; see Smith and Smith, 1996 for a review) as the fungus may continue
to draw carbohydrates from the plant, despite the fact that the plant
can obtain P directly from the soil. In addition, whereas plants often
show the greatest positive growth response at low phosphorus conditions,
some studies have found the opposite (e.g., Li et al., 2005).

The plant-mycorrhizal interaction may also shift between a
mutualistic and parasitic interaction depending on the life stage of the
plant. When plants are young, the cost of the carbohydrate drain by the
fungus may be greater than the benefit received by the plant from
increased phosphorus availability, resulting in growth depression (e.g.,
Bethlenfalvay et al., 1982; Koide, 1985). Age of the plant was also
important for Hyacinthoides non- scripta, as the mycorrhizal associated
shifted from facultative to obligate with plant age (Merryweather and
Fitter, 1995). The relative cost-benefit of the association may also
shift seasonally, as the mycorrhizae are costly to Erythronium
americanum in the fall but beneficial in the spring (Lapointe and
Molard, 1997). These shifts among parasitic, neutral, and mutualistic
interactions over time indicate the importance of longer term
experiments that incorporate all stages of the plant life cycle.
Incorporating reproductive output and fitness estimates is of particular
importance for understanding the ecological and evolutionary
implications of plant-mycorrhizal associations.

Relatively few studies have included plant reproductive output,
particularly for nonagricultural species (see Koide, 2000; Varga, 2010
for reviews). Although increases in plant size often result in greater
fecundity, the effects are not consistent (Jones and Smith, 2004); and
extrapolating linearly from short term growth experiments could lead to
incorrect assumptions about future fecundity. In addition, whereas the
presence of mycorrhizae can increase seed output, the effect on
reproduction can vary with environment (e.g., Carey et al., 1992), plant
genotype (e.g., Bryla and Koide, 1990), and plant density (e.g., Koide
et al., 1994). For species that can reproduce vegetatively, mycorrhizae
have the potential to significantly alter allocation to clonal growth
(e.g., Streitwolf-Engle et al., 1997) and to shift relative resource
allocation between vegetative propagules and flowers (e.g., Scagel and
Schreiner, 2006) thus further complicating assumptions about mycorrhizal
effects on fitness. If mycorrhizae alter the allocation patterns to
different reproductive modes, it could have important implications for
the genetic and spatial structure of the population.

In this study I investigated the effect of phosphorus level on the
response of Allium vineale (wild garlic or onion grass) to mycorrhizal
colonization across all life stages of the plant. Specifically, I asked
how variation in P level affects the plant-fungal association, including
affects on overall plant size, resource allocation patterns, and
fecundity. By using P levels within the same range found in the field
from which the plants originated, the responses measured here give an
indication of whether variation in nutrient availability in the field
could cause the effect of mycorrhizae to shift along the
mutualistic-parasitic continuum (Johnson et al., 1997). Allium vineale
reproduces via three kinds of propagules, sexually produced seeds,
underground asexual offsets, and aerial asexual bulbils; thus I was able
to test for shifts in the allocation of resources to different
reproductive modes as well as overall effects of mycorrhizal
colonization on plant fecundity.

METHODS

Study species and site description.--Allium vineale L. (Liliaceae)
is a naturalized introduced species commonly found in fields and along
roadsides from Michigan to Georgia (Radford et al., 1968). It is a
winter perennial that sprouts in early autumn, grows throughout the
winter and dies back in Jun. Plants produce a new bulb each year, as
well as up to three types of propagules. Offsets are underground asexual
propagules analogous to the cloves of domesticated garlic. They are the
largest of the propagule types (mean mass = 217 mg, ranging from 40-600
mg), and, unlike the bulb that resprouts in the fall, they may remain
dormant in the soil for up to 5 y (Stritzke and Peters, 1972). As the
leaves die back in early summer, A. vineale produces a scape with an
inflorescence containing bulbils (asexual reproduction), flowers (sexual
reproduction), or a combination of both. Seeds and bulbils ripen in
early fall and disperse to a mean distance of 34 cm from the parent
(Ronsheim, 1994). Seeds weigh approximately 1 mg each, whereas bulbils
range from 5-60 mg (mean mass = 19.8 mg; Ronsheim, 1996). A. vineale
roots are coarse (0.25-1 mm in diameter), rarely branched, and colonized
by arbuscular mycorrhizal (AM) fungi in the field (Richens, 1947; M.
Ronsheim, pers. obs.).

The field site is on the Vassar College Ecological Preserve in
Dutchess County, New York. The field was previously used for agriculture
but was abandoned in the late 1950s and is currently maintained by
mowing every few years. It is dominated by Bromus inermis Leyss., Galium
mollugo L., Poa pratensis L., Comus racemosa Lam., Rhus radicans L., and
Solidago sp. Allium vineale is a common species (reproductive
individuals were present in 21 out of 49 one [m.sup.2] quadrats
sampled). P availability ranges from 39-155 [micro]g x [g.sup-1] readily
extractable P, with a mean of 95.4 [micro]g x g-1 dw (all soil analyses
conducted by A&L Eastern Agricultural Laboratories, Inc., Richmond
VA, using methods from A. L. Page, 1982, see below).

Experimental design.--Allium vineale bulbils were collected from
the field and planted in pots either with or without mycorrhizae at six
levels of P fertilization. Bulbils were collected from 13 parents and
randomly assigned to each treatment. Mycorrhizal pots were inoculated
with roots of Plantago lanceolata L. and Bromus inermis collected from
the field site that were heavily colonized by AM fungi. Because A.
vineale plants in the field are dormant and have little active root
material when the bulbils are mature, roots from these two co-occurring
species were used; and thus the specific mycorrhizae present may be
different from those found on A. vineale roots in the field. The roots
were surface sterilized in a 5% bleach solution, chopped into 1-2 cm
lengths, thoroughly mixed, and then approximately 20 mg (wet mass) was
added to the soil. The bulbils were then weighed and planted slightly
above the inoculum.

Plants were fertilized with Hoagland's solution modified to
contain one of six different P concentrations (0, 50, 100, 150, 200, or
250 [micro]g x L P) once a week for 6 wk (19 replicates per treatment,
[n.sub.tota] = 228). Twenty-five ml of fertilizer were added during the
first two fertilizations and 20 ml during the remaining four
fertilizations. The soil was a 1:1 mixture by volume of heat pasteurized
field soil and sterile sand to improve drainage. At the start of the
experiment the soil mixture had a pH of 5.5, with 1.8% organic matter
and 91 [micro]g x g-1 dw readily available P (all soil analyses
conducted by A&L Eastern Agricultural Laboratories, Inc., Richmond
VA, using methods from A. L. Page, 1982). Soil pH was measured at 1:1
soil to water solution, percent organic matter was determined on dried
screened soil, and total available P was determined using the Bray P-1
procedure. The pH of the soil used in this experiment is slightly higher
than the original field soil (pH 5.2) and the soil organic matter is
lower than the original field soil (4.0% organic matter). Soil was
collected from several pots from each P treatment at the end of the
experiment, and the level of readily extractable P across the six
treatments was 89, 118, 138, 172, 201, and 193 [micro]g x g-1 dw
respectively. The lack of difference between the last two P treatments
may be due to the high sand content of the soil resulting in the
leaching out of the additional added P.

Because Allium vineale roots produce few branches and tend to grow
vertically in the soil, circular pots 3.8 cm in diameter, and 21 cm deep
were used (Conetainers, Stuewe, and Sons, Inc., Corvallis, Oregon, USA).
The plants were randomly arranged within trays and placed in a growth
chamber set to average Oct. temperature and light conditions (12.5 h
daylight, daytime T = 16 C, nighttime T = 7 C).

The first of three harvests was done 1 mo after planting (n = 72),
the second after 6 mo (n = 62), and the final harvest was done after 15
mo (n = 70). Of the original 228 bulbils planted, 24 did not germinate
and were not included in the analyses. Failure to germinate was random
across treatments. The dry mass of shoots and roots and the percent AM
fungal colonization was determined at 1 and 6 too. For those plants that
had a subset of their roots examined for AM fungal colonization rates
(all mycorrhizal plants and a subset of nonmycorrhizal plants), the
total dry root mass was estimated using a regression of the wet and dry
root mass. Percent AM fungi colonization was determined using the
gridline intersect method described by Giovannetti and Mosse (1980) on
roots stained using the procedure described by Grace and Stribley
(1991). No AM fungal colonization was observed in plants from the
noninoculated treatment.

The final harvest (15 mo) was done when the plants entered dormancy
and all their roots and leaves had died back, leaving only underground
bulbs, offsets and aerial reproductive stalks. Total dry biomass, bulb
mass, and the number and mass of offsets, bulbils and flowers were
recorded.

Data analysis.--Data were analyzed using the GLM procedure in SAS
(SAS, 1986) with initial mass of the bulbils as a covariate. Response
variables reported here include total biomass at 1, 6, and 15 mo,
root:shoot allocation and percent root colonization at 1 and 6 mo, and
the mass of bulbs, offsets, bulbils, and flowers at 15 mo. The number
and mean mass of bulbils and offsets per plant were also analyzed.
Biomass data from the first two harvests were log transformed to improve
normality. Proportions were arcsine square root transformed. Post hoc
comparisons of least squares means were made using Tukey's
criterion to correct for multiple comparisons.

Differences in reproductive allocation patterns among plants in the
mycorrhizal and P soil treatments were tested using two-way multivariate
analyses of variance with profile contrasts using ranked data (Repeated
Profile option in GLM procedure in SAS; Morrison, 1976; Ronsheim and
Bever, 2000). Profile analysis tests whether the slopes of lines
connecting the means of each reproductive character (bulb, offset,
bulbil, and flower biomass) differ between the soil treatments, allowing
all four characters to examined simultaneously. For example, a
significant Mycorrhizae * Profile interaction would indicate that
mycorrhizal and nonmycorrhizal plants differ in their allocation to
different reproductive modes. The significance of these interactions was
tested using Wilk's Lambda criterion because it is derived from a
likelihood ratio approach (SAS, 1986); however, tests using
Pillai's Trace and Hotelling-Lawley Trace gave similar results.
Additional analyses of pairs of reproductive characters (e.g., bulbils
vs. flowers) gave the same results as the overall profile analysis and
will not be presented here.

After 6 mo, there was no significant effect of mycorrhizal
treatment on biomass (P = 0.104, Fig. 1b) although there was a trend for
mycorrhizal plants to be larger at lower P levels (Myc * P interaction;
P = 0.087). The effect of P addition on total plant biomass did not
change over this time period, as plant biomass was significantly larger
with higher P levels (P < 0.001).

After 15 mo a significant Mycorrhizae * P interaction was seen for
total biomass, with mycorrhizal plants being significantly larger than
nonmycorrhizal plants at 0 and 50 [micro]g x g-1 P (P < 0.001, Fig.
1c). At higher P levels, there was no difference in the size of
mycorrhizal and nonmycorrhizal plants. Thus, a significant mycorrhizal
benefit in terms of increased total biomass was demonstrated only at
lower P fertilization levels and only after 15 mo of growth.

Reproduction.--Mycorrhizal and nonmycorrhizal plants had
significantly different responses to increased level of P fertilization
in the production of underground asexual offsets (Myc * P interaction, P
= 0.004). The total biomass of offsets produced by mycorrhizal plants
did not vary with P treatment. In contrast, the total mass of offsets
for nonmycorrhizal plants growing at the two lowest P levels was
significantly smaller than for nonmycorrhizal plants at higher P levels,
as well as being significantly smaller than the total biomass of offsets
produced by mycorrhizal plants at 0 [micro]g x g-1 added P. Whereas
there was no significant variation in offset number for any of the
treatments (offset number; Myc P = 0.270, P P = 0.550, Myc * P P =
0.282), the average mass of offsets was significantly smaller for
nonmycorrhizal plants at low P levels relative to nonmycorrhizal plants
at higher P levels and relative to mycorrhizal plants at 0 [micro]g x
g-1 added P (Myc * P interaction, P = 0.001, Fig. 3a). Thus, the
presence of mycorrhizae in low P treatments increased the relative size
but not number of underground asexual propagules.

A similar pattern was seen for the total biomass of bulbils, which
did not vary for mycorrhizal plants but was significantly lower for
nonmycorrhizal plants growing with 0 [micro]g x g-1 added P relative to
other nonmycorrhizal plants growing at 200 and 250 [micro]g x g-1 added
P and to mycorrhizal plants at 0 [micro]g x g-1 added P (Myc * P
interaction, P < 0.001). Most of this variation in total bulbil
biomass was due to an increase in the number of bulbils rather than a
change in the average size of individual bulbils (number of bulbils, Myc
* P, P = 0.006, Fig. 3b). At 0 [micro]g x g-1 added P none of the
nonmycorrhizal plants produced an aerial reproductive stalk, and at 50
[micro]g x g-1 added P only two of six nonmycorrhizal plants produced an
aerial reproductive stalk. In all other treatments either five of six or
six of six plants produced an aerial reproductive stalk.

Mycorrhizal plants produced larger bulbs than nonmycorrhizal plants
across all levels of P added (P < 0.001, Fig. 3c). Mycorrhizal plants
also produced more flowers than nonmycorrhizal plants (P = 0.016).
Plants in both treatments produced few flowers, with nonmycorrhizal
plants producing a mean of 1.6 flowers and mycorrhizal plants producing
a mean of 2.6 flowers.

The profile analyses indicate that resource allocation to the
different propagule types (bulb, offsets, bulbils, and flowers) was
marginally different for mycorrhizal and nonmycorrhizal plants (Myc *
Profile interaction, P = 0.059). However, most of this variation was due
to the difference in bulb size for mycorrhizal vs. nonmycorrhizal
plants, and there was no evidence for a significant shift in the
relative allocation to offset, bulbil or flower production for
mycorrhizal vs. nonmycorrhizal plants. In particular, there was no
difference in allocation to flowers vs. bulbils, with an overall 13.7%
flowers/(flowers + bulbils). There was no significant difference in
allocation to different propagule types among the phosphorus treatments
(P * Profile interaction, P = 0.122), nor was a significant interaction
effect found (Myc * P * Profile, P = 0.065).

[FIGURE 2 OMITTED]

[FIGURE 3 OMITTED]

DISCUSSION

For Allium vineale plants, the impact of mycorrhizal infection
varies significantly with life stage. Young plants (1 mo) experience a
significant growth depression, which disappears as the plants grow (6
mo), and finally switches to a positive association when the plants
reach their reproductive stage (15 mo). Mycorrhizal induced growth
depressions are often assumed to be a result of the balance between
benefits and costs of the plant- fungal association, in which fungal
demands for C from the plant are not balanced by the benefits of P
transport to the plant (see Jones and Smith, 2004), and this negative
effect can persist through the life of the plant. The growth depression
seen in this study clearly does not fall into this category, as the
presence of mycorrhizae at later life stages results in significantly
larger plants and more reproductive output at lower P levels. Given
recent evidence for P uptake even when there is no growth advantage
(e.g., Li et al., 2008; Smith et al., 2009), young mycorrhizal A.
vineale plants may have been receiving and accumulating P in their bulb
that was then used when the carbon assimilating potential of the plants
reached a threshold level. Without information on P uptake and transport
by the fungus and the plant, coupled with information on rates of C
assimilation and transport by the plant, we cannot determine what, if
any, role the C-P cost-benefit balance played in the growth depression
and its reversal in this study, underscoring the need for further
mechanistic investigation of early growth depression (see review by
Smith et al., 2009).

Mycorrhizal induced growth depressions can also occur at higher
levels of nutrient availability when P is not limiting, as plants
experience the carbon cost of the fungus but without the benefit of
increased available P (Johnson et al., 1997; Jones and Smith, 2004). In
this study we found no evidence for environmental parasitism of the
plant by mycorrhizae at high levels of P, although the plant-fungal
association does shift from a relative benefit at lower P levels to
neutral at higher P levels. In particular, there was no effect of
variation in P level on overall size of mycorrhizal plants at 15 mo or
on their reproductive output. As seen in some other species (e.g.,
Hepper, 1983; Jensen, 1983; Thomson et al., 1986; Schroeder and Janos,
2004; see also Smith and Smith, 1996), the percent of Allium vineale
roots colonized by mycorrhizae was significantly lower at higher levels
of P after 6 mo of growth. It is possible that this reduction in
colonization by the mycorrhizal fungus lowers the cost of the
association for the plant, thereby resulting in an overall neutral
effect rather than a negative effect on total plant biomass (Johnson et
al., 1997).

In contrast to mycorrhizal plants, P level had a significant effect
on nearly all traits measured for nonmycorrhizal plants, indicating that
nonmycorrhizal plants in the low P treatment were limited by P
availability. Nonmycorrhizal plants in higher P treatments allocated
less resources to roots, were significantly larger at 15 mo, and
produced more bulbils and larger offsets. At the lowest P level none of
the nonmycorrhizal plants produced an aerial scape, whereas at higher P
levels their total size, root:shoot allocation and reproductive output
was not significantly different from mycorrhizal plants. Plants often
allocate more to roots when belowground resources such as P are limiting
(Ericsson, 1995; Jones and Smith, 2004). The fact that mycorrhizal
plants had larger bulbs and higher reproductive rates at maturity may be
at least partly due to the fact they were able to allocate less
resources to roots at low P levels.

Information on the impact of mycorrhizae on plant reproduction is
clearly important for developing an ecological/evolutionary perspective
on plant-mycorrhizal dynamics. In general, species with positive
vegetative responses also have positive reproductive responses, but this
pattern is not universal and varies with environment and plant genotype
(e.g., Scagel and Schreiner, 2006) and mycorrhizal inoculum (e.g.,
Oliveria et al., 2006). In this study, mycorrhizal plants at lower P
levels had higher fecundity than nonmycorrhizal plants, as they produced
more bulbils and larger offsets. In addition, the presence of
mycorrhizae resulted in larger bulbs at all levels of P. As larger bulbs
produce greater numbers of flowers and bulbils the following year
(Ronsheim, 1997), this increase in bulb size is likely to translate into
greater lifetime fitness, and this benefit may be increased further if
the specific fungal associates of Allium vineale are present. Whereas
mycorrhizal plants also produce, on average, an additional flower
relative to nonmycorrhizal plants, it is difficult to determine if this
increase would have a significant effect on overall fitness. Both seed
set (mean of 0.7 seeds/flower in the field, M. Ronsheim, pers. obs.) and
seedling survival (Ronsheim, 1996) are relatively low in the field thus
the impact of one additional flower on fitness and overall population
dynamics is likely to be small.

The presence of mycorrhizae did not shift the relative allocation
of resources to different reproductive modes (bulbils vs. offsets vs.
flowers). Thus, the relative allocation of resources to sexual vs.
asexual reproduction as well as to above vs. belowground asexual
reproduction is unaffected by mycorrhizal status. Mycorrhizal Allium
vineale plants did produce more bulbils and larger offsets than
nonmycorrhizal plants but only at lower P levels. A previous study
demonstrated that A. vineale plants produce more bulbils and larger
offsets with increased nutrient availability (Ronsheim and Bever, 2000)
thus it is likely that the increase in reproductive output found in this
study is also consequence of improved nutritional status, specifically
an increase in P availability. Selection for aerial dispersal ability
could be one factor that would favor producing a greater number of
smaller aerial bulbils rather than fewer larger ones, but the potential
advantages of increasing the size vs. number of underground offsets is
unknown.

The P concentrations used in this experiment (89-201 [micro]g x
g-1) reflect the upper range in P availability found in the field from
which these plants were collected (39-155 [micro]g x g-1). The results
from this study indicate that, within this population of Allium vineale,
spatial heterogeneity in P in the field is likely to result in the
plant-mycorrhizal association ranging from beneficial to neutral in its
effect on plant growth and reproduction. Recent work by Johnson et al.
(2010) demonstrates the presence of local co-adaptation in plant- AM
fungal symbioses, resulting in a geographic mosaic that maximizes
benefits in P limited soil and minimizes costs in P rich soil. Whether
variation within a field in nutrient availability and in the relative
benefit of mycorrhizae could result in small scale local adaptation of
plant-fungal communities is unknown.

In summary, these results emphasize the importance of long term
studies that include all life stages of the plant, especially those
relating to reproduction and overall fitness. In addition, spatial
variation in nutrient availability in the field has the potential to
shift the overall effect of mycorrhizae from beneficial to neutral, with
greater benefits found in microsites with lower phosphorus levels.
Finally, whereas colonization by mycorrhizae does increase overall bulb
size and thus potentially the long term fitness of the plant, it does
not affect how resources are allocated among propagule types.

Acknowledgments.--I would like to thank K. Sharma, B. Gumbs, and S.
Anderson for their help with this project and L. Christenson and two
anonymous reviewers for comments on the manuscript.